FN CC>SS/CC>SS embryos display growth retardation and abnormal vascular

To investigate the reasons for the embryonic lethality of FNCC>SS/CC>SS embryos, embryos derived from heterozygous intercrosses were isolated at different stages of development to compare mutant with control littermates. The morphology of mutant embryos isolated at E8.5 was undistinguishable from wild-type littermates; they exhibited well developed head folds, 5 – 6 somite pairs and a properly developed neural tube (Fig 3.20 A, B). First indications of impaired development became apparent at

~E9.0. All FNCC>SS/CC>SS embryos at E9.5 were developmentally retarded when compared with their wild-type littermates (Fig. 3.20 E, F). They were markedly reduced in size, displayed a retarded posterior development and rarely developed beyond 20 pairs of somites. Another hallmark of mutant embryos at E9.5 was the appearance of oedema, indicated by a noticeably enlarged pericardial sac (Fig 3.20 F, H), which suggested that the FN mutation affected the development of the cardiovascular system.

At E10.5, the growth arrest affecting mutant embryos became more evident and in some mutant hearts a cardiac-looping defect was observed (Fig 3.20 H).

Fig 3.20. Whole mount images of wild-type and FNCC>SS/CC>SS embryos. A and B. Side view of E8.5 embryos. Wild-type and mutant embryos are comparable in size and exhibit well developed allantoises. C and D. Dorsal view of E8.5 embryos. Note that both, wild-type and mutant, exhibit 5 pairs of somite blocks. The insets shows properly shaped somite blocks at higher magnification. E and F. Side view of E9.5 embryos. FNCC>SS/CC>SS embryos are severely growth retarded and underlie a growth arrest at the 20 - 25 somite pair stage. Note that mutants show oedema in the heart, as indicated by an enlarged pericardial sac (F and H). G and H. Side view of E10.5 embryos. The growth arrest in FNCC>SS/CC>SS embryos leads to a pronounced difference in size as compared to the wild-type littermates. The arrowhead points to the markedly effused pericardial sac and the noticeable malformed heart, which is observed in most mutant embryos

(H). al, allantois; fl, fore limb bud; h, heart; pl, placenta; s, somites; ys, yolk sac; Scale bars:

(D,F) 500 µm; (H) 1 mm.

The effused pericardial sac and the growth arrest observed in FNCC>SS/CC>SS embryos beyond E9.0 suggested that the cardiovascular development was affected by the mutation. Clear indications of abnormal vascular development were also visible in FNCC>SS/CC>SS yolk sacs by E9.5. Compared to control yolk sacs, mutant yolk sacs were anemic and lacked a distinct branching network of vessels, which became even more pronounced by E10.5 (Fig 3.21.A). Some regions on the mutant yolk sac exhibited only small and disorganized vessels, whereas other regions completely lacked vessels.

Furthermore, the presence of red blood cells in the mutant yolk sac cavity indicated leakiness of present small vessels, and suggested that the circulation would be impaired.

To characterize these defects in more detail, the morphology of the vessel architecture in embryonic and extra-embryonic tissue was examined by means of immunohistochemistry. To this end, immunofluorescence stainings for the endothelial marker PECAM-1 (CD31) were performed on whole-mount yolk sacs. The analysis revealed that mutant yolk sacs failed to remodel their primitive vascular plexus by angiogenesis (Fig 3.21.C), which starts beyond E8.5 (Risau 1997) and results in formation of individual branched vessels through endothelial cell outgrowth and splitting of existing vessels.

The observed abnormal vascular development in the yolk sacs of FNCC>SS/CC>SS embryos suggested that other parts of the extra-embryonic tissue, such as the ectoplacental plate, may also be affected. To test this, paraffin sections of E10.5 placentas were stained for the endothelial marker Endomucin. At this stage of development, chorioallantoic fusion has been accomplished and fetal blood vessels derived from the extra-embryonic tissue have already invaded the maternal labyrinthine layer of the placenta in control mice.

Intermingling of embryonic and maternal vessels in this layer is essential for proper exchange of gases and nutrition between the two circulations. The analysis of Endomucin stained ectoplacental plates revealed that the allantoic blood vessels of FNCC>SS/CC>SS mutants completely failed to invade the maternal labyrinthine layer. The mutant vessels residing in the embryonic part of the chorioallantoic plate were dilated or sometimes collapsed (Fig 3.21.F).

Fig 3.21. Abnormal vascular development in the extra-embryonic tissue of FNCC>SS/CC>SS mice.

A. Gross morphology of whole-mount yolk sacs of mutant embryos compared to those of wild-type littermates at E10.5. FNCC>SS/CC>SS yolk sacs exhibit severe defects in vascular development.

Arrowheads show a defined and organized vessel structure in the wild-type yolk sac (left), whereas the mutant yolk sac exhibits severely dilated and leaky blood vessels with bleedings into the yolk sac tissue (right arrowhead and inset). B and C. Yolk sacs at E9.5, stained with an antibody against the endothelial marker PECAM-1. In contrast to the large vessels developed in wild-type yolk sacs, the primitive vascular plexus in mutant yolk sacs is not remodelled into an organized network of larger vessels (C). E and F. Paraffin sections of placentas at E10.5 stained with an antibody against the endothelial marker endomucin. Note that embryonic vessels of FNCC>SS/CC>SS embryos fail to invade and sprout properly into the maternal labyrinthine layer (F) The arrowhead marks a vessel of collapsed appearance. ca, chorioallantoic plate; ll, labyrinthine layer; pl, placenta; ys, yolk sac; Scale bars: (A) 1 mm; (C, F) 100 µm.

To analyse the vessel architecture in embryonic tissue, the vascular system of whole E9.5 embryos was also stained with antibodies against endothelial markers. Similar to the observations made in extra-embryonic tissues, whole-mount staining of embryos

with endomucin antibodies revealed a markedly impaired capability of FNCC>SS/CC>SS

embryos to develop a properly branched vascular system. In contrast to control embryos at this stage, FNCC>SS/CC>SS embryos exhibited dilated small vessels of fragile appearance and notably reduced lumina. Some regions of the mutant embryo - particular in the head and in intersomitic regions of the posterior trunk - showed only a weak expression of endomucin, suggesting an abnormal endothelial differentiation (Fig 3.22.A, C). Furthermore, immunofluorescence stainings with an antibody against PECAM-1 revealed that mutant vessels were irregularly shaped and showed numerous dilations. Some regions of the mutant embryo proper completely lacked a discernable organisation of vessels into a branched network (Fig 3.22.E).

Fig 3.22. Abnormal vascular development in FNCC>SS/CC>SS embryos. A. Image of whole-mount embryos at E9.5, stained with antibodies against endomucin. Mutant embryos (right) display a profoundly impaired vessel architecture as compared to control embryos (left). B and C. Higher magnification of embryo head-regions (from A). Note that FNCC>SS/CC>SS embryos form fragile, disoriented vessels with markedly reduced lumina (C). D and E. Immunofluorescence image of the head region of E9.5 embryos stained with antibodies against PECAM-1. Note the dilated vessels with markedly diverse diameters in FNCC>SS/CC>SS embryos (E), compared to control embryos (D). Arrowheads in D and E mark regions of normally developed vessels in the wild-type (D), and a severly disoriented vessel architecture in the head of a mutant embryo (E), respectively. Scale bars: (A) 500 µm; (C) 250 µm; (E) 100 µm

Together, the expression of FN-monomers results in a growth arrest at the 20-25 somite stage with an abnormal vascular development in both embryonic and extra-embryonic tissues. Defects in vascular development may explain embryonic lethality at the time-point when the embryo starts to become dependent on placental supply. In the case of FNCC>SS/CC>SS embryos, however, the growth is arrested around E9.5 (Fig 3.20.F) when

the embryo is not yet dependent on placental exchange of nutrients (Cross, Werb et al.

1994).